A. Technical Field
This invention relates to a transfusable composition of cells produced by ex vivo growth processes. More particularly, it relates to transfusable compositions that are universally compatible, perform the same physiologic function as red blood cells (xe2x80x9crbcsxe2x80x9d or xe2x80x9cerythrocytesxe2x80x9d) from human donors, and supplement or replace the current practice of transfusing red blood cells from human donors.
B. Background Art
1. Current Blood-Banking Practice
Blood transfusion is a critical component of current medical practice. Each year there are about 12 million donation units transfused in the United States. Of these, 3.2 million units are used to treat chronic anemia, 8 million units are used to treat surgical blood loss and about 750,000 units are used to treat traumatic blood loss. As practiced currently, blood transfusion involves drawing blood from a presumed healthy donor, mixing it with an anticoagulant/preservative, testing it for immunologic reactivity (typing), testing it for known infectious agents (currently eight) and storing it for administration to suitable recipient/patient. Prior to administration to the recipient, a sample of the donor blood is tested in combination with a sample of the recipient""s blood to determine immunologic compatibility (cross-matching). After establishing compatibility, the donated blood is intravenously infused into the recipient. Despite the testing precautions, blood transfusions pose life-threatening risks to the recipient. Epstein, Increasing Safety of Blood Transfusions, Amer. Red Cross (1992).
Although there are recognized therapeutic applications for white blood cells, platelets and other components of blood, e.g. coagulation factors, plasma proteins. etc., the predominate use for blood transfusions is based on the oxygen-carrying ability of the red blood cells. A unit of whole blood is 500 milliliters or approximately one pint. Adults have from about 9 to about 12 pints of blood in their bodies. Blood donated for transfusion is typically composed of 40-50% (by volume) red blood cells and 50-60% plasma (liquid component). One milliliter of blood typically contains 4 to 5 billion rbcs, which is equivalent to 2.0 to 2.5 trillion rbcs per donation unit.
Blood typing is the process by which red blood cells are tested to determine which cell surface antigens are present and absent. It is standard blood-banking practice to test routinely for the A, B, and D (Rh) antigens and to test for other antigens only in selected cases. When a person lacks a particular red blood cell antigen, his or her plasma may contain an antibody to that antigen. Some antibodies (e.g. anti-A and anti-B) are naturally occurring and are expected to be present. Other antibodies are unexpected. They usually result from a challenge to the immune system by exposure to foreign blood cells, such as through transfusion or pregnancy. A small percentage of persons carry antibodies to certain blood cell antigens without prior exposure.
Antibody reactions with antigens present on infused red cells often result in a serious adverse clinical event known as a xe2x80x9ctransfusion reaction.xe2x80x9d With the exception of the ABO blood group system, the kinds of antibodies that cause transfusion reactions are found almost exclusively in persons who have had prior transfusions or pregnancies. Antibodies in the patient""s plasma are detected in screening studies using panels of red cells containing known antigens.
Blood group antigens are glycoprotein or glycolipid structures on the surface of the red cell membrane and can be removed or modified in potency by using various enzymes, e.g. glycosidases, proteases, etc. Red cell antigens vary in their immunogenicity and prevalence.
In routine blood-banking practice, only ABO typing and Rh grouping are performed. A crossmatch procedure is performed as a final check for compatibility.
Crossmatching is performed by mixing the donor""s cells with the recipient""s serum, with and without enhancing agents, and observing for agglutination (clumping) or lysis (destruction) of the red cells. Either of these events signals that a problem antibody to an antigen on the donor""s cells is present in the recipient""s plasma and signals the likelihood of a transfusion reaction.
The frequency and identity of problem antibodies/blood types have been established (in order of decreasing frequency):
1. Anti-D (with or without anti-C or anti-E);
2. Anti-Lea and anti-Leb (alone or together);
3. Anti-K;
4. Anti-E anti-P1;
5. Anti-c, Anti-cE, Anti-Fya, Anti-M;
6. Anti-jKa, Anti-S;
7. Anti-Ce, anti-E;
8. Anti-jKb, anti-N, Anti-s. anti-Fyb.
Because of a variable and uncertain supply (often critical shortages), the potential for transmitting blood-borne disease, and the risk of immunologic incompatibilities and transfusion reactions, there is a recognized and substantial need to supplement or replace the current practice of transfusing blood from human donors.
2. Ex Vivo Cell Culture
Mature blood cells result from the growth and differentiation of hematopoietic cells. Hematopoietic cells are generated from pluripotent stem cells, which can both self-renew and give rise to hematopoietic progenitor cells. Hematopoietic progenitor cells include lymphoid, mixed-lineage colony-forming units (CFU-Mix), granulocyte/macrophage colony-forming units (CFU-GM), erythrocyte burst-forming units (BFU-E), and megakaryocyte burst-forming units (BFU-meg). These progenitors, in turn, give rise to mature blood cells. (Koller et al., Biotechnol. Bioeng. 42: 477 (1993).
Primitive hematopoietic cells reside in the bone arrow of normal adults, where they mature into functional blood cells and are released into the peripheral circulation. The bone marrow is a complex environment consisting of stem, progenitor and mature hematopoietic cells, along with accessory cells and molecules (in extracellular matrix) which are necessary to maintain the process of hematopoiesis. Accessory cells and the extracellular matrix mediate the differentiation and proliferation of hematopoietic cells by producing growth factors and by direct cell contact. Bone marrow, thus, is a natural source of hematopoietic cells at various stages of differentiation, as well as a source of accessory cells. Immature hematopoietic cells can also be harvested from peripheral blood, with or without stimulation by growth factors, using leukopharesis. This process separates the nucleated cells of interest from red blood cells and plasma. Another source of immature hematopoietic cells is umbilical cord blood. Bone marrow has been the traditional source of hematopoietic cells for transplantation therapies, although peripheral blood progenitor cell transplants have also proven useful. Demuynck, et al., Ann. Hematol. 71: 29 (1995).
Cancer chemotherapy often results in severe damage to hematopoietic progenitor cells. As a result, the patient is susceptible to infection and bleeding. A therapy for this damage involves transplantation of bone marrow or hematopoietic cells. The loss of hematopoietic activity, due to damage by chemotherapy, can be offset by infusing hematopoietic cells into the patient after chemotherapy. Thus, there has been much interest and in harvesting and expanding hematopoietic progenitor cells in ex-vivo cell cultures.
Ex vivo expansion of hematopoietic progenitor cells can decrease the amount of the initial harvest necessary for successful engraftment and, most importantly, can improve the transplant outcome by allowing more cells to be transplanted. Such expansion supplements transplants with mature progenitors and speeds the recovery of mature white cells and platelets, which in turn fight infection and control bleeding, respectively. In addition, ex vivo expansion also allows the use of a single hematopoietic cell harvest for repeated transplants over an extended period of time. Collins. et al., Curr. Opin. in Biotechnol., 7: 223 (1996).
Ex vivo expansion of hematopoietic cells includes the following prerequisites: First, cells positive for the CD34 antigen are extracted because these cells are presumed to represent the most primitive hematopoietic cells. Second, the extract is further purified for CD34+ cells having other antigens associated with the desired progeny. Third, selected cells are added to growth media, with or without exogenous growth factors and with or without serum. Fourth, the cell/growth medium is incubated in a bioreactor to control the environment for expansion and/or the differentiation of primitive or mature hematopoietic cells. Finally, the expanded and differentiated cells are harvested and purified.
While all of these prerequisites are routinely practiced for providing hematopoietic cells for bone marrow transplantation, a transfusable composition of oxygenating cells produced by the ex vivo culture of hematopoietic cells is still not available. In fact, the current state of the art of hematopoietic cell culture is directed away from erythropoiesis (production of red blood cells) in favor of producing infection-fighting white blood cells (leukopoiesis) and platelets to control bleeding.
3. Bioreactors
Bioreactors are systems that provide an environment for the growth, expansion and differentiation of cells in culture. Bioreactor systems have been described as static, perfusion, and stirred suspension systems. Static culture systems, such as culture well plates and flasks are the most popular means for expanding hematopoietic cells. Koller, et al., Biotechnol. Bioeng., 50: 505 (1996). However, these systems present significant difficulties in controlling nutrient concentrations and waste product concentrations and cannot support high cell densities. Although exchanging as much as 50% of the medium has been effective in increasing total cell production, exchange of all the medium does not increase culture performance, presumably due to the removal of coincidental growth factors or the disruption of cell-to-cell interactions.
Perfusion systems have been developed to exchange the culture medium continuously without mechanically damaging cells or reducing the concentration of critical factors. Sandstrom, et al., Biotechnol. Bioeng., 50: 493 (1996). Such perfusion systems have shown greater cell expansion than static systems and have successfully expanded human bone marrow progenitor cells, CFU-GM and BFU-E. Cameron, R. B., U.S. Pat. Nos. 5,599,705 and 5,811,301, describes in vitro production of what are said to be transfusable differentiated, universally compatible human blood cells by expansion of pluripotent hematopoietic stem cells in perfusion type bioreactors, such as those having ceramic matrix cores, hollow capillary fibers or protein-coated microspheres.
Stirred vessels have been suggested as the system of choice for many mammalian cell culture applications, because they offer advantages in sampling, data collection and control of medium conditions. Zandstra, et al., Biotechnology, 12:909 (1994). Nevertheless, the adaptation of these stirred systems to hematopoietic cell cultures has proven to be a challenge, because of the uncertain effect of stirring on the disruption of cell-to-cell associations common to these types of cultures.
4. Perfluorocarbons
Because cell cultures involve living processes, respiration at the cellular level must be supported. Respiration for mammalian cells involves the exchange of essentially two gases, oxygen and carbon dioxide. Depending on the specific configuration of the bioreactor, gas transport to each cell growing in the bioreactor, can be impeded by the cell growth density (cell accumulation) and the oxygen-carrying capacity and distribution of the medium. Koller, et al., Biotechnol. Bioeng., 42:477 (1993). Perfluorocarbons (xe2x80x9cPFCsxe2x80x9d), particularly perfluorodecalin and perfluorooctane, have been demonstrated to carry as much as four times the amount of oxygen as water. Also, their oxygen transport capacity is directly proportional to their concentration and p02. Riess, I. G., Vox Sang., 61:225 (1991).
Because PFCs dissolve gases, such as oxygen and carbon dioxide, to a greater extent than aqueous culture media, they aid in transfer of oxygen to and removal of carbon dioxide from cells. They also are very compatible with mammalian cells. These features obviate the need to provide excessive gas to the medium for oxygenation of cells and waste gas removal. The use of PFCs as a means optimally to regulate oxygenation or regulate mixing kinetics in bioreactors have not been utilized for production of transfusable differentiated blood cells obtained by in vitro.
PFCs are typically introduced into aqueous media as emulsions. Not only do the emulsions serve to transport dissolved gases, they can also increase the viscosity of the media, thereby presenting an alternative method to control the rheology or mixing kinetics of the cell culture. For optimal control, the viscosity should be high enough to form a protective shield against shear impulses, but not so high as to impose a detrimental barrier to mass transfer for supply of nutrients or removal of waste products. Because ex vivo erythropoiesis occurs in different phases of development, each requiring unique growth factors, gas mixtures, and degrees of perfusion, no single bioreactor design has been shown to support the entire process from expansion of the primitive hematopoietic cell through expansion and differentiation of erythroid (referring to red blood cells) progenitors to the maturation of mature transfusable erythrocytes.
In accordance with this invention, a process for producing a transfusable, oxygenating composition of red blood cells comprises:
(a) expanding a culture of purified primitive hematopoietic cells in a first bioreactor in the presence of growth factors and under conditions that promote self renewal of such cells to increase their numbers without substantial differentiation into committed progenitor cells, thereby producing an expanded culture of purified primitive hematopoietic cells;
(b) effecting differentiation of the primitive hematopoietic cells by culturing said expanded culture of purified primitive hematopoietic cells in a second bioreactor in the presence of differentiation factors and under conditions that promote the differentiation of the primitive hematopoietic cells into erythroid progenitor cells;
(c) producing mature erythrocytes by culturing said erythroid progenitor cells in a third bioreactor in the presence of maturation factors and under conditions that promote the maturation of progenitor erythroid cells into mature erythrocytes; and
(d) harvesting said mature erythrocytes and formulating them into transfusable oxygenating composition.
In a preferred embodiment, perfluorocarbons are employed in the culture media used in one or more of the bioreactors. The perfluorocarbons promote transfer of respiratory gases to and from the cells in such culture media.
As discussed below, the various bioreactors may be different vessels arranged in tandem or may be the same vessel with changes in media, added factors and/or conditions. Further, additional bioreactors may be employed in the process, for example, for maintenance and/or storage of cell cultures.
The process of this invention provides a source of transfusable red blood cells that may be universally compatible and free of the risk of contamination with is infectious agents. Because the process is based on in vitro expansion of primitive hematopoietic stem cells obtained, for example, from bone marrow explants, the need for reliance on blood donations is diminished.